Cooperation between the two heads of smooth muscle myosin is essential for full activation of the motor function by phosphorylation.

The motor function of smooth muscle myosin (SmM) is regulated by phosphorylation of the regulatory light chain (RLC) bound to the neck region of the SmM heavy chain. It is generally accepted that unphosphorylated RLC induces interactions between the two heads and between the head and the tail, thus inhibiting the motor activity of SmM, whereas phosphorylation of RLC interrupts those interactions, thus reversing the inhibition and restoring the motor activity to the maximal value. One assumption of this model is that single-headed SmM is fully active regardless of phosphorylation. To re-evaluate this model, we produced a number of SmM constructs with coiled coils of various lengths and examined their structure and regulation. With these constructs we identified the segment in the coiled-coil key for the formation of a stable double-headed structure. In agreement with the current model, we found that the actin-activated ATPase activity of unphosphorylated SmM increased with shortening of the coiled-coil. However, contrary to the current model, we found that the actin-activated ATPase activity of phosphorylated SmM decreased with shortening coiled-coil and only the stable double-headed SmM was fully activated by phosphorylation. These results indicate that single-headed SmM is neither fully active nor fully inhibited. Based on our findings, we propose that cooperation between the two heads is essential, not only for the inhibition of unphosphorylated SmM, but also for the activation of phosphorylated SmM.

Ablation of smooth muscle caldesmon affects the relaxation kinetics of arterial muscle.

Smooth muscle caldesmon (h-CaD) is an actin- and myosin-binding protein that reversibly inhibits the actomyosin ATPase activity in vitro. To test the function of h-CaD in vivo, we eliminated its expression in mice. The h-CaD-null animals appeared normal and fertile, although the litter size was smaller. Tissues from the homozygotes lacked h-CaD and exhibited upregulation of the non-muscle isoform, l-CaD, in visceral, but not vascular tonic smooth muscles. While the Ca(2+) sensitivity of force generation of h-CaD-deficient smooth muscle remained largely unchanged, the kinetic behavior during relaxation in arteries was different. Both intact and permeabilized arterial smooth muscle tissues from the knockout animals relaxed more slowly than those of the wild type. Since this difference occurred after myosin dephosphorylation was complete, the kinetic effect most likely resulted from slower detachment of unphosphorylated crossbridges. Detailed analyses revealed that the apparently slower relaxation of h-CaD-null smooth muscle was due to an increase in the amplitude of a slower component of the biphasic tension decay. While the identity of this slower process has not been unequivocally determined, we propose it reflects a thin filament state that elicits fewer re-attached crossbridges. Our finding that h-CaD modulates the rate of smooth muscle relaxation clearly supports a role in the control of vascular tone.

Modular structure of smooth muscle Myosin light chain kinase: hydrodynamic modeling and functional implications.

Smooth muscle myosin light chain kinase (smMLCK) is a calcium-calmodulin complex-dependent enzyme that activates contraction of smooth muscle. The polypeptide chain of rabbit uterine smMLCK (Swiss-Prot entry P29294) contains the catalytic/regulatory domain, three immunoglobulin-related motifs (Ig), one fibronectin-related motif (Fn3), a repetitive, proline-rich segment (PEVK), and, at the N-terminus, a unique F-actin-binding domain. We have evaluated the spatial arrangement of these domains in a recombinant 125 kDa full-length smMLCK and its two catalytically active C-terminal fragments (77 kDa, residues 461-1147, and 61 kDa, residues 461-1002). Electron microscopic images of smMLCK cross-linked to F-actin show particles at variable distances (11-55 nm) from the filament, suggesting that a well-structured C-terminal segment of smMLCK is connected to the actin-binding domain by a long, flexible tether. We have used structural homology and molecular dynamics methods to construct various all-atom representation models of smMLCK and its two fragments. The theoretical sedimentation coefficients computed with HYDROPRO were compared with those determined by sedimentation velocity. We found agreement between the predicted and observed sedimentation coefficients for models in which the independently folded catalytic domain, Fn3, and Ig domains are aligned consecutively on the long axis of the molecule. The PEVK segment is modeled as an extensible linker that enables smMLCK to remain bound to F-actin and simultaneously activate the myosin heads of adjacent myosin filaments at a distance of >or=40 nm. The structural properties of smMLCK may contribute to the elasticity of smooth muscle cells.

The motor activity of myosin-X promotes actin fiber convergence at the cell periphery to initiate filopodia formation.

Filopodia are actin-rich fingerlike protrusions found at the leading edge of migrating cells and are believed to play a role in directional sensing. Previous studies have shown that myosin-X (myoX) promotes filopodia formation and that this is mediated through its ability to deliver specific cargoes to the cell periphery (Tokuo, H., and M. Ikebe. 2004. Biochem Biophys. Commun. 319:214-220; Zhang, H., J.S. Berg, Z. Li, Y. Wang, P. Lang, A.D. Sousa, A. Bhaskar, R.E. Cheney, and S. Stromblad. 2004. Nat. Cell Biol. 6:523-531; Bohil, A.B., B.W. Robertson, and R.E. Cheney. 2006. Proc. Natl. Acad. Sci. USA. 103:12411-12416; Zhu, X.J., C.Z. Wang, P.G. Dai, Y. Xie, N.N. Song, Y. Liu, Q.S. Du, L. Mei, Y.Q. Ding, and W.C. Xiong. 2007. Nat. Cell Biol. 9:184-192). In this study, we show that the motor function of myoX and not the cargo function is critical for initiating filopodia formation. Using a dimer-inducing technique, we find that myoX lacking its cargo-binding tail moves laterally at the leading edge of lamellipodia and induces filopodia in living cells. We conclude that the motor function of the two-headed form of myoX is critical for actin reorganization at the leading edge, leading to filopodia formation.

The globular tail domain of myosin Va functions as an inhibitor of the myosin Va motor.

The actin-activated ATPase activity of full-length mammalian myosin Va is well regulated by Ca2+, whereas that of truncated myosin Va without the C-terminal globular tail domain (GTD) is not. Here, we have found that exogenous GTD is capable of inhibiting the actin-activated ATPase activity of GTD-deleted myosin Va. A series of truncated constructs of myosin Va further showed that the entire length of the first coiled-coil (coil-1) of the tail domain is critical for GTD-dependent regulation of myosin Va and that deletion of 58 residues from the C-terminal end of coil-1 markedly hampered regulation. Negative staining electron microscopy revealed that GTD-deleted myosin Va formed a "Y"-shaped structure, which was converted to a triangular shape, similar to the structure of full-length myosin Va in the inhibited state, by addition of exogenous GTD. In contrast, the triangular shape was not observed when the C-terminal 58 residues of coil-1 were deleted, even in the presence of exogenous GTD. Based on these results, we propose a model for the formation of the inhibited state of myosin Va. GTD binds to the C-terminal end of coil-1. The neck-tail junction of myosin Va is flexible, and the long neck enables the head domain to reach the GTD associated with the end of coil-1. Once the head interacts with the GTD, the triangular inhibited conformation is stabilized. Consistent with this model, we found that shortening of the neck of myosin Va by two IQ motifs abolished the regulation by GTD, whereas regulation was partially restored by shortening of coil-1 by an amount comparable to that of the two IQ motifs.

The calcium- and zinc-responsive regions of calreticulin reside strictly in the N-/C-domain.

Calreticulin (CRT) is a chaperone of the endoplasmic reticulum. We dissected CRT into its two structural domains, N-/C-domain and P-domain, to identify its metal ion-responsive region. For this, we constructed bacterial expression systems for the N-/C-domain (1-180 fused by a linker to 290-400) and P-domain (189-280). Circular dichroism (CD) studies showed that calcium ions increased tertiary packing and thermal stability of apo N-/C-domain, whereas zinc ions had a strong destabilizing effect. Interestingly, neither calcium nor zinc ions altered the structural properties of apo P-domain. These results indicate that the calcium- and zinc-responsive regions reside strictly in the N-/C-domain. Analysis of thermal denaturation curves of CRT, N-/C-domain, and P-domain suggested a structural role for the P-domain in CRT. Rotary shadowing electron microscopy (EM) analysis of CRT and calnexin provided convincing evidence for their structural relatedness. This analysis also revealed that apo P-domain adopts various curved shapes suggesting conformational flexibility. EM images of apo N-/C-domain revealed objects having wide gaps suggesting weak interactions between the N- and C-domains. This is consistent with the larger size of apo N-/C-domain on the gel filtration column. Our studies provide a framework for correlating the structural organization of CRT with its metal ion-responsive region.

Mechanoenzymatic characterization of human myosin Vb.

There are three isoforms of class V myosin in mammals. While myosin Va has been studied well, little is known about the function of other myosin V isoforms (Vb and Vc) at a molecular level. Here we report the mechanoenzymatic function of human myosin Vb (HuM5B) for the first time. Electron microscopic observation showed that HuM5B has a double-headed structure with a long neck like myosin Va. V(max) and K(actin) of the actin-activated ATPase activity of HuM5B were 9.7 +/- 0.4 s(-)(1) and 8.5 +/- 0.1 microM, respectively. K(actin) and K(ATP) of the actin-activated ATPase activity were significantly higher than those of myosin Va. ADP markedly inhibited the ATPase activity. The rate of release of ADP from acto-HuM5B was 12.2 +/- 0.5 s(-)(1), which was comparable to the V(max) of the actin-activated ATPase activity. These results suggest that ADP release is the rate-limiting step for the actin-activated ATPase cycle; thus, HuM5B is a high duty ratio myosin. Consistently, the actin gliding velocity (0.22 +/- 0.03 microm/s) remained constant at a low motor density. The actin filament landing assay revealed that a single HuM5B molecule is sufficient to move the actin filament continuously, indicating that HuM5b is a processive motor.

Characterization and function of MYPT2, a target subunit of myosin phosphatase in heart.

Characterization of cardiac MYPT2 (an isoform of the smooth muscle phosphatase [MP] target subunit, MYPT1) is described. Several features of MYPT2 and MYPT1 were similar, including: a specific interaction with the catalytic subunit of type 1 phosphatase, delta isoform (PP1cdelta); interaction of MYPT2 with the small heart-specific MP subunit; interaction of the C-terminal region of MYPT2 with the active form of RhoA; phosphorylation by Rho-kinase at an inhibitory site, Thr646 and thiophosphorylation at Thr646 inhibited activity of the MYPT2-PP1cdelta complex. MYPT2 activated PP1cdelta activity, using light chains from smooth and cardiac muscle, by reducing K(m) and increasing k(cat). The extent of activation (k(cat)) was greater than for MYPT1 and could reflect distinct N-terminal sequences in the two MYPT isoforms. Adenovirus-mediated gene transfer of MYPT2 and PP1cdelta reduced the phosphorylation level of cardiac light chains following stimulation with A23187. Overexpression of MYPT2 and PP1cdelta blocked the angiotensin II-induced sarcomere organization in cultured cardiomyocytes. Electron microscopy indicated locations of MYPTs, at, or close to, the Z-line, the A band and mitochondria. Similarity of the two MYPT isoforms suggests common enzymatic mechanisms and regulation. Cardiac myosin is a substrate for the MYPT2 holoenzyme, but the Z-line location raises the possibility of other substrates.

Ca2+-induced activation of ATPase activity of myosin Va is accompanied with a large conformational change.

We succeeded in expressing the recombinant full-length myosin Va (M5Full) and studied its regulation mechanism. The actin-activated ATPase activity of M5Full was significantly activated by Ca(2+), whereas the truncated myosin Va without C-terminal globular domain is not regulated by Ca(2+) and constitutively active. Sedimentation analysis showed that the sedimentation coefficient of M5Full undergoes a Ca(2+)-induced conformational transition from 14S to 11S. Electron microscopy revealed that at low ionic strength, M5Full showed an extended conformation in high Ca(2+) while it formed a folded shape in the presence of EGTA, in which the tail domain was folded back towards the head-neck region. Furthermore, we found that the motor domain of myosin Va folds back to the neck domain in Ca(2+) while the head-neck domain is more extended in EGTA. It is thought that the association of the motor domain to the neck inhibits the binding of the tail to the neck thus destabilizing a folded conformation in Ca(2+). This conformational transition is closely correlated to the actin-activated ATPase activity. These results suggest that the tail and neck domain play a role in the Ca(2+) dependent regulation of myosin Va.

M20, the small subunit of PP1M, binds to microtubules.

Myosin light chain phosphatase (PP1M) is composed of three subunits, i.e., M20, MBS, and a catalytic subunit. Whereas MBS is assigned as a myosin binding subunit, the function of M20 is unknown. In the present study, we found that M20 binds to microtubules. The binding activity was revealed by cosedimentation of M20 with microtubules and binding of tubulin to M20 affinity resin. Green fluorescent protein (GFP)-tagged M20 (M20-GFP) was expressed in chicken primary smooth muscle cells and COS-7 cells and was used as a probe for studying the association between M20 and microtubules in living cells. M20-GFP was localized on filamentous structures in both cell types. Colocalization analysis revealed that M20-GFP colocalized with tubulin. Treatment with nocodazole, but not cytochalasin B, abolished the filamentous structure of M20-GFP. These results indicate that M20-GFP associates with microtubules in cells. Microinjection of rhodamine-tubulin into the M20-expressing cells revealed that incorporation of rhodamine-tubulin into microtubules was significantly facilitated by microtubule-associated M20. Consistent with this result, M20 enhanced the rate of tubulin polymerization in vitro and produced elongated microtubules. These results suggest that M20 has a microtubule binding activity and plays a role in regulating microtubule dynamics.

Hsp72 and stress kinase c-jun N-terminal kinase regulate the bid-dependent pathway in tumor necrosis factor-induced apoptosis.

The major inducible heat shock protein Hsp72 has been shown to protect cells from certain apoptotic stimuli. Here we investigated the mechanism of Hsp72-mediated protection from tumor necrosis factor (TNF)-induced apoptosis of primary culture of IMR90 human fibroblasts. Hsp72 temporarily blocked apoptosis in response to TNF and permanently protected cells from heat shock. An Hsp72 mutant (Hsp72 Delta EEVD) with a deletion of the four C-terminal amino acids, which are essential for the chaperone function, blocked TNF-induced apoptosis in a manner similar to that of normal Hsp72 but did not inhibit heat shock-induced death. Therefore, the chaperone activity of Hsp72 is dispensable for suppression of TNF-induced apoptosis but is required for protection from heat shock. In fibroblasts derived from Bid knockout mice, similar temporal inhibition of TNF-induced apoptosis was seen. In these cells neither normal Hsp72 nor Hsp72 Delta EEVD conferred additional protection from apoptosis, suggesting that Hsp72 specifically affects Bid-dependent but not Bid-independent apoptotic pathways. Furthermore, both normal Hsp72 and Delta Hsp72EEVD inhibited Bid activation and downstream events, including release of cytochrome c, activation of caspase 3, and cleavage of poly-ADP-ribose polymerase. Both Hsp72 and Delta Hsp72EEVD blocked activation of the stress kinase c-jun N-terminal kinase (JNK) by TNF, and specific inhibition of JNK similarly temporarily blocked Bid activation and the downstream apoptotic events. These data strongly suggest that in TNF-induced apoptosis, Hsp72 specifically interferes with the Bid-dependent apoptotic pathway via inhibition of JNK.